The human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine kinase receptor protein. HER2 gene amplification and receptor overexpression, which occur in 15–20% of breast cancer patients, are important markers for poor prognosis. Moreover, HER2-positive status is considered a predictive marker of response to HER2 inhibitors including trastuzumab and lapatinib. Therefore, reliable HER2 determination is essential to determine the eligibility of breast cancer patients to targeted anti-HER2 therapies. In this chapter, we aim to illustrate important aspects of the HER2 receptor as well as the molecular consequences of its aberrant constitutive activation in breast cancer. In addition, we will present the methods that can be used for the evaluation of HER2 status at different levels (protein, RNA, and DNA level) in clinical practice.
- breast neoplasm
- tyrosine kinase receptor
- molecular oncology
- HER2 status
- HER2 inhibitors
Breast cancer is the most frequently diagnosed cancer among women worldwide, affecting over 1.5 million women each year. In 2015, it is estimated that worldwide 500,000 women have died from this malignancy, which represents 15% of all cancer-related deaths among women .
It is now well recognized that breast cancer comprises a heterogeneous group of diseases in term of differentiation and proliferation, prognosis and treatment. Over the past decades, microarray-based gene expression studies have allowed the identification of breast cancer intrinsic subtypes [2, 3, 4]. One of these subtypes is the so-called human epidermal growth factor receptor 2 (HER2)-enriched subtype. HER2 is a transmembrane tyrosine kinase receptor . This protein is encoded by the
HER2 plays a significant role in breast cancer pathogenesis. It is therefore essential to understand the biology of this receptor in order to better treat HER2-positive breast cancer patients. Evaluation of HER2 status in breast cancer specimens raises several technical considerations. In the last decades, several methods have been developed for HER2 assessment. In this article, we will review important aspects of the HER2 biology and its relevance in breast cancer and present the techniques that are used in clinical practice for the determination of HER2 status in breast cancer specimens.
2. HER2 biology and methods of assessment of HER2 status
2.1. HER2 receptor
The HER2 receptor is a 185 kDa transmembrane protein that is encoded by the
2.1.1. HER2 structure and function
HER2 belongs to the epidermal growth factor receptor (EGFR) family. This family is composed of four HER receptors: human epidermal growth factor receptor 1 (HER1) (also termed EGFR), HER2, human epidermal growth factor receptor 3 (HER3), and human epidermal growth factor receptor 4 (HER4) .
HER family members are transmembrane receptor tyrosine kinases. Tyrosine kinases are enzymes that carry out tyrosine phosphorylation, namely the transfer of the γ phosphate of adenosine triphosphate (ATP) to tyrosine residues on protein substrate .
HER receptors share a similar structure. They are composed of an extracellular domain (ECD), a transmembrane segment and an intracellular region . The ECD domain is divided into four parts: domains I and III, which play a role in ligand binding, and domains II and IV, which contain several cysteine residues that are important for disulfide bond formation . The transmembrane segment is composed of 19–25 amino acid residues. The intracellular region is composed of a juxtamembrane segment, a functional protein kinase domain (with the exception of HER3 that lacks tyrosine kinase activity  and must partner with another family member to be activated ), and a C-terminal tail containing multiple phosphorylation sites required for propagation of downstream signaling . The catalytic domain contains the ATP binding pocket, a conserved site essential to ATP binding .
HER receptors are activated by both homo- and heterodimerization, generally induced by ligand binding . This suggests that HER receptor family has evolved to provide a high degree of signal diversity . The cellular outcome produced by HER receptors activation depends on the signaling pathways that are induced, as well as their magnitude and duration, which are influenced by the composition of the dimer and the identity of the ligand .
Several growth factor ligands interact with the HER receptors . HER1 receptor is activated by six ligands: epidermal growth factor (EGF), epigen (EPG), transforming growth factor α (TGFα), amphiregulin, heparin-binding EGF-like growth factor, betacellulin and epiregulin. HER3 and HER4 receptors bind neuregulins (neuregulin-1, neuregulin-2, neuregulin-3, and neuregulin-4). HER2 is a co-receptor for many ligands and is often transactivated by EGF-like ligands, inducing the formation of HER1-HER2 heterodimers. Neuregulins induces the formation of HER2-HER3 and HER2-HER4 heterodimers . However, no known ligand can promote HER2 homodimer formation, implying that no ligand can bind directly to HER2 .
The structural basis for receptor dimerization has been elucidated in recent years through crystallographic studies [31, 32]. Dimerization is mediated by the dimerization arm, a region of the extracellular region of HER receptors. While in its inactivated state the dimerization arm of EGFR, HER3 and HER4 is hidden, ligand binding induces a receptor conformational change leading to exposure of the dimerization arm . In contrast to the other three HER receptors, the dimerization arm of the HER2 receptor is permanently partially exposed, thus permitting its dimerization even if the HER2 receptor lacks ligand-binding activity .
Interaction between the dimerization arms of two HER receptors promotes the formation of a stable receptor dimer in which the kinase regions of both receptors are closed enough to permit transphosphorylation of tyrosine residues, i.e. the transfer of a phosphate group by a protein kinase to a tyrosine residue in a different kinase molecule [33, 34]. The first member of the dimer mediates the phosphorylation of the second, and the second dimer mediates the phosphorylation of the first .
The phosphorylation of specific tyrosine residues following HER receptor activation and the subsequent recruitment and activation of downstream signaling proteins leads to activation of downstream signaling pathways promoting cell proliferation, survival, migration, adhesion, angiogenesis and differentiation . The Phosphatidylinositol 3′-kinase (PI3K)-Akt pathway and the Ras/Raf/MEK/ERK pathway (also known as extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway) are the two most important and most extensively studied downstream signaling pathways that are activated by the HER receptors [5, 36]. These downstream signaling cascades control cell cycle, cell growth and survival, apoptosis, metabolism and angiogenesis [37, 38]. Signaling from HER receptors is then terminated through the internalization of the activated receptors from the cell surface by endocytosis. Internalized receptors are then either recycled back to the plasma membrane (HER2, HER3, HER4) or degraded in lysosomes (HER1) [39, 40].
HER heterodimers produce more potent signal transduction than homodimers. This can be explained by the fact that heterodimerization provides additional phosphotyrosine residues necessary for the recruitment of effector proteins . Heterodimerization follows a strict hierarchical principle with HER2 representing the preferred dimerization and signaling partner for all other members of the HER family . HER2 seems to function mainly as a co-receptor, increasing the affinity of ligand binding to dimerized receptor complexes [42, 43]. HER2 has the strongest catalytic kinase activity  and HER2-containing heterodimers produce intracellular signals that are significantly stronger than signals generated from other HER heterodimers . The HER2-HER3 heterodimer in particular exhibits extremely potent mitogenic activity through the stimulation of the PI3K/Akt pathway, a master regulator of cell growth and survival . Furthermore, HER2 containing heterodimers have a slow rate of receptor internalization, which results in prolonged stimulation of downstream signaling pathways . HER2 can also be activated by complexing with other membrane receptors, such as Insulin-like growth factor I receptor (IGF-1R) .
2.1.2. Consequences of constitutive HER2 receptor activation
Whereas in normal cells the activity of tyrosine kinases is a tightly controlled mechanism, in cancer cells, alterations in tyrosine kinases—overexpression of receptor tyrosine kinase proteins, amplification or mutation in the corresponding gene, abnormal stimulation by autocrine growth factors loop or delayed degradation of activated receptor tyrosine kinase—lead to constitutive kinase activation and therefore to aberrant cellular growth and proliferation [34, 47]. Constitutive activation of HER1, HER2, HER3, IGF-1R, Fibroblast growth factor receptor (FGFR), c-Met, Insulin Receptor (IR), Vascular Endothelial Growth Factor Receptor (VEGFR), Jak kinases and Src have been associated with human cancer [34, 48, 49, 50, 51, 52].
Several ways of aberrant activation of HER receptors have been described, including ligand binding, molecular structural alterations, lack of the phosphatase activity, or overexpression of the HER receptor .
In HER2-positive tumors, receptor overexpression has been identified as the mechanism of HER2 activation. The increased amount of cell surface HER2 receptors associated with HER2 overexpression leads to increased receptor-receptor interactions, provoking a sustained tyrosine phosphorylation of the kinase domain and therefore constant activation of the signaling pathways. HER2 overexpression also enhances HER2 heterodimerization with HER1 and HER3  resulting in an increased activation of the downstream signaling pathways. It has also been shown that HER2 overexpression leads to enhanced HER1 membrane expression and HER1 signaling activity through interference with the endocytic regulation of HER1 [54, 55, 56]. While HER1 undergoes endocytic degradation after ligand-mediated activation and homodimerization, HER1-HER2 heterodimers evade endocytic degradation in favor of the recycling pathway [57, 58], resulting in increased HER1 membrane expression and activity [55, 56, 59].
It has also been reported that HER2 overexpression enhances cell proliferation through the rapid degradation of the cyclin-dependent kinase (Cdk) inhibitor p27 and the upregulation of factors that promote cell cycle progression, including Cdk6 and cyclins D1 and E .
Several methods have been developed for the assessment of HER2 status in breast cancer specimens, at the protein level, DNA level, and RNA level. Here below, we present some of the existing techniques that are used for the HER2 determination in clinical practice.
2.2. Methods for the evaluation of HER2 status in breast cancer specimens
2.2.1. HER2 status evaluation at the protein level
188.8.131.52. Immunohistochemistry (IHC)
IHC allows the evaluation of the HER2 protein expression in formalin-fixed, paraffin-embedded (FFPE) tissues using specific antibodies directed against the HER2 receptor protein . HER2 receptor is then visualized with the chromogen 3,3′-diaminobenzidine tetrahydrochloride (DAB) resulting in a brownish membranous staining. Several commercially available diagnostic tests for the determination of HER2 expression have been approved by the FDA: the HercepTest™ kit (DAKO, Glostrup, Denmark), the InSite™ HER2/neu kit (clone CB11; BioGenex Laboratories, San Ramon, CA), the Pathway™ kit (clone 4B5; Ventana Medical Systems, Tucson, AZ), and the Bond Oracle HER2 IHC System (Leica Biosystems, Newcastle, UK).
By this method, it is possible to estimate the number of cells showing membranous staining in the tissue section as well as the intensity of the staining . Membranous staining in the invasive component of specimen is scored on a semi-quantitative scale. According to the American Society of Clinical oncology (ASCO) and the College of American Pathologists (CAP) recommendations for HER2 testing in breast cancer published in 2013, HER2 expression is scored as 0 (no staining or weak/incomplete membrane staining in ≤10% of tumor cells), 1+ (weak, incomplete membrane staining in >10% of tumor cells), 2+ (strong, complete membrane staining in ≤10% of tumor cells or weak/moderate and/or incomplete membrane staining in >10% of tumors cells) or 3+ (strong, complete, homogeneous membrane staining in >10% of tumor cells) . In clinical practice, HER2 immunohistochemical status is evaluated as negative if the immunohistochemical score is 0 or 1+, equivocal is the score is 2+, and positive if the score is 3+. Patients with a positive HER2 status at the IHC are eligible for targeted therapy with HER2 inhibitors. The IHC 2+ category is considered borderline and confirmatory testing using an alternative assay (fluorescence
IHC is an easy and relatively inexpensive method . However, this technique can be affected by numerous factors, including warm/cold ischemic time , delay and duration of fixation , and antibody used [66, 67]. Moreover, since the interpretation of results is based on semiquantitative scoring, this technique is prone to interobserver variability and therefore to substantial discrepancies in the IHC results, particularly for cases scoring 2+ .
184.108.40.206. Enzyme-linked immunosorbent assay (ELISA)
As mentioned before, HER2 receptor is composed of an extracellular domain (ECD), a transmembrane domain, and an intracellular domain with tyrosine kinase activity. The HER2 ECD can be cleaved from the HER2 full-length receptor through matrix metalloproteases and released into the serum . HER2 ECD levels present in serum can be measured using an enzyme-linked immunosorbent assay (ELISA). HER2 ECD is detected using two antibodies that recognize two specific epitopes of the antigen. Several commercially available ELISA assays received FDA approval: the automated ELISA assay Immuno-1 (Siemens Healthcare Diagnostics, Tarrytown, NY), the manual ELISA assay (Siemens Healthcare Diagnostics) in 2000, and the automated ELISA assay ADVIA Centaur (Siemens Healthcare Diagnostics) in 2003 .
Although some studies suggest that HER2 ECD levels measured in patient’s serum could be used as a biomarker for the monitoring of the disease course and the response of the patient to therapy, the clinical use of the ELISA assay for the evaluation of the HER2 ECD has not yet been widely implemented [71, 72]. This is mainly due to the fact that studies that analyzed the association between HER2 ECD levels and prognostic and predictive factors in breast cancer patients reported conflicting results, depending on which cutoff value was considered or which assay was used .
ELISA is an easy and fast method. In addition, given that HER2 ECD can be measured directly in serum, ELISA can be used to monitor the dynamic changes of HER2 status following treatment or over the course of the disease progression . Results obtained by ELISA, however, might not be reliable if the serum samples are from patients under treatment, as trastuzumab present in the patient’s serum might compete with the two antibodies used in the assay.
2.2.2. HER2 status evaluation at the DNA level
in situhybridization (FISH)
The FISH technique is a cytogenetic technique that uses fluorescent probes to target specific DNA sequences in FFPE tissue samples . FISH is effectuated either as a single-color assay (
Three FISH assay kits have been approved by the FDA for the determination of the
According to the 2013 ASCO/CAP guidelines, a case is evaluated as amplified when the mean
Although still matter of debate, several researchers consider FISH as being more accurate and reliable than IHC in the assessment of HER2 status in breast cancer specimens [77, 78, 79, 80]. In addition, given that DNA is more stable than protein, preanalytical factors have less impact on assay results compared with IHC . Although the FISH technique yields results that are considered more objective and quantitative than immunohistochemical scoring [73, 82], this method is nine times more time-consuming  and three times more expensive compared with IHC . In addition, costly equipment is required for signal detection . The FISH assay can be interpreted only by well-trained personnel, as distinguishing invasive breast cancer from breast carcinoma
Moreover, fluorescence signal counting is time consuming. To overcome this limitation, image analysis software for the automated assessment of fluorescence signals has been developed. Several investigators have reported an excellent concordance between HER2/CEP17 ratios calculated through manual counting and those obtained with automated image analysis system [86, 87, 88]. Some image analysis systems has been approved by the FDA for the automated determination of
in situhybridization (ISH) methods
Given that FISH technology have some limitations, alternative ISH methods have been developed for the assessment of
Since visualization is achieved using other reactions than fluorescence-labeled probe, signals can be evaluated using a standard bright-field microscope, allowing the simultaneous analysis of
in situhybridization (CISH)
CISH allows the visualization of target genes in breast cancer tissue sections through peroxidase enzyme-labeled probes . The single-color CISH assay (SPOT-Light HER2 CISH kit; Life Technologies, Carlsbad, CA), and the dual-color CISH assay (HER2 CISH PharmDx kit; Dako) received FDA approval in 2008 and 2011, respectively .
With the single-color CISH assay, only the absolute
The dual-color CISH assay allows the simultaneous visualization of the HER2 and CEP17 probes on the same slide . HER2 probes are visualized using a chromogen (green as example), whereas CEP17 probes are visualized using another chromogen (red as example). Slides are then counterstained with hematoxylin. Results obtained by dual-color CISH are reported as dual-color FISH .
The CISH assay is twice cheaper  and 1.2 times faster  comparatively to FISH. Furthermore, since the CISH assay allows an easier identification of the invasive component compared with FISH, evaluation of CISH signals is less time-consuming than FISH [82, 94]. In addition, tumor heterogeneity is promptly recognizable, even at low magnification . Moreover, the dual-color assay can be performed on an automated slide stainer, improving the reproducibility of the assay . However, the assessment of
in situhybridization (SISH)
SISH is an automated enzyme metallography assay, in which an enzyme reaction is used to selectively deposit metallic silver from solution at the reaction site to produce a black staining . All steps of the assay are performed on the Ventana BenchMark XT automated slide stainer [98, 99]. HER2 and chromosome 17 analysis is performed on sequential slides [98, 99]. As previously mentioned, HER2 and CEP17 probes are visualized through the process of enzyme metallography. During the process, silver precipitation is deposited in the nucleus, and HER2 or CEP17 signals are visualized as black dots within cell nuclei . Similar to the FISH assay,
Given that the SISH assay is fully automated, this technique is six times faster to perform than the FISH assay . In addition, black SISH signals are easier to evaluate compared with other bright-field ISH techniques [100, 101]. However, to correct for chromosome 17 aneusomy, the hybridization of a further section is required for separate assessment of CEP17 copy number .
220.127.116.11. Bright-field double ISH (BDISH)
Bright-field double ISH (BDISH) or dual-color
This technique is very pertinent especially for cases displaying chromosome 17 aneusomy or intratumoral heterogeneity, as it allows the simultaneous visualization of both HER2 and CEP17 probes on the same slide . Furthermore, as the HER2 signals and CEP17 signals differ in color and size (HER2 black spots are smaller than CEP17 red spots), both signals can be distinguished from each other, even though they colocalize within cell nuclei . Moreover, since this assay is completely automated, results are available within 6 h, in addition of being more reproducible, as risk of human errors are diminished . The BDISH assay presents the same disadvantages as CISH and SISH.
18.104.22.168. Instant-quality FISH (IQFISH) and automated
Recently, new FISH assays have been developed for the evaluation of
2.2.3. HER2 status evaluation at the RNA level
22.214.171.124. Polymerase chain reaction (PCR)-based assays
Polymerase chain reaction (PCR) is a technique used for the detection of DNA samples through the exponential amplification of target DNA sequences.
Reverse transcription PCR (RT-PCR) assay allows the quantification of mRNA and can be used for the evaluation of HER2 expression in breast cancer specimens in both FFPE and frozen tissues [106, 107]. Extracted mRNA is at first reverse transcribed into complementary DNA (cDNA). cDNA is then measured by quantitative PCR (qPCR). The relative quantitation of
RT-PCR has a large dynamic range, in addition of being a quantitative method. PCR results, however, are often associated with false-negative results due to dilution of amplified tumor cells with surrounding nonamplified stromal cells [108, 109]. In addition, the evaluation of HER2 status at the mRNA level by RT-PCR using FFPE tissues can be problematic, as mRNA integrity can be damaged by several factors, including tissue fixation and storage time .
HER2 is a prognostic marker in breast cancer. HER2 overexpression and
In addition, HER2-positive status is considered a predictive marker of response to HER2-targeted drugs, including trastuzumab and lapatinib . Considering the clinical and economic implications of targeted anti-HER2 treatments, reliable HER2 test results are essential. False negative results would deny the patients access to the potential benefits of trastuzumab, whereas false positive results would expose patients to the potential cardiotoxic side effects of this expensive agent without experiencing any therapeutic advantages .
Although several techniques have obtained FDA approval for the HER2 assessment in breast cancer specimens, the ASCO/CAP guidelines recommend performing IHC or ISH methods to determine HER2 status in breast cancer. The optimal method for evaluating HER2 status in breast cancer specimens, however, is still matter of debate, since each method is characterized by its own advantages and disadvantages. Therefore, emphasis must be put on standardization of procedures and quality control assessment of already existing methods. Also, development of new accurate assays should be promoted. Moreover, large clinical trials are needed to identify the technique that most reliably predicts a positive response to HER2 inhibitors.
DF received doctoral fellowships from the Fonds de recherche du Québec—Santé (FRQS) and the Laval University Cancer Research. CD is a recipient of the Canadian Breast Cancer Foundation-Canadian Cancer Society Capacity Development award (award #703003) and the FRQS Research Scholar.
Conflict of interest
The authors have no conflicts of interests to declare.
The authors have no other declarations.
Acronyms and abbreviations
|HER2||Human epidermal growth factor receptor 2|
|GRB7||Growth factor receptor bound protein 7|
|ESR1||Estrogen Receptor 1|
|FDA||Food and Drug Administration|
|EGFR||Epidermal growth factor receptor|
|FISH||Fluorescence in situ hybridization|
|ERBB2||erb-b2 receptor tyrosine kinase 2|
|HER3||Human epidermal growth factor receptor 3|
|HER4||Human epidermal growth factor receptor 4|
|EGF||Epidermal growth factor|
|TGFα||Transforming growth factor α|
|ERK||Extracellular signal-regulated kinase|
|MAPK||Mitogen-activated protein kinase|
|FGFR||Fibroblast growth factor receptor|
|VEGFR||Vascular Endothelial Growth Factor Receptor|
|ASCO||American Society of Clinical Oncology|
|CAP||College of American Pathologists|
|ELISA||Enzyme-linked immunosorbent assay|
|CEP17||Chromosome enumeration probe 17|
|ISH||in situ hybridization|
|CISH||Chromogenic in situ hybridization|
|SISH||Silver-enhanced in situ hybridization|
|BDISH||Bright-field double ISH|
|PCR||polymerase chain reaction|
|RT-PCR||Reverse transcription PCR|